Methods, compositions, and kits for improving pancreatic beta cell viability and treating diseases or conditions related to beta cell destruction

ABSTRACT

The present disclosure provides novel methods for increasing β-cell viability in islets by delivering RLIP76 polypeptides or GSTA4 polypeptides, or a combination thereof; or RLIP76 polynucleotides or GSTA4 polynucleotides, or a combination thereof, to the islets. The disclosure also provides novel methods for treating a disease or condition in a subject, such as type 1 diabetes mellitus, by delivering RLIP76 polypeptides or GSTA4 polypeptides, or a combination thereof; or RLIP76 polynucleotides or GSTA4 polynucleotides, or a combination thereof, to islets and transplanting the islets into the subject to treat the disease or condition. Kits and compositions including RLIP76 polypeptides or GSTA4 polypeptides, or a combination thereof; or RLIP76 polynucleotides or GSTA4 polynucleotides, or a combination thereof, are also provided to increase β-cell viability.

PRIORITY CLAIM

This application is a continuation application of U.S. application Ser.No. 14/659,568 filed Mar. 16, 2015, which application claims the benefitof U.S. Provisional Application No. 61/953,725 filed Mar. 14, 2014, eachof which is hereby incorporated by reference as if fully set forthherein.

SEQUENCE LISTING

This application contains a Sequence Listing, which was submitted inASCII format via EFS-Web, and is hereby incorporated by reference in itsentirety. The ASCII copy, created on May 23, 2018, is named54435-8133US2_SequenceListing.txt and is 21 KB in size.

BACKGROUND

Human islet transplant frequently fails due to apoptosis and necrosisoccurring in the cells prior to transplantation. Successfultransplantation is hampered by poor survival of transplanted insulinsecreting beta (β) cells contained in the islets. Oxidative stress andother stresses are a major cause of decrease in cell or tissue viabilityand loss or insulin-secretion function prior to transplantation. Thesuccess-rate of human islet-cell transplantation is highly dependent onthe number of viable insulin-secreting cells transplanted.

Islet transplantation has been successful in treating many patients withtype 1 diabetes mellitus. However, this therapeutic procedure is limitedbecause of the chronic shortage of cadaveric pancreata. Therefore,developing methods to increase islet mass by stimulating theproliferation of islet cells in-vitro would have a significant impactfor expansion of insulin producing β-cells for transplant applications.Pancreatic β-cells are relatively susceptible to the damaging effects ofoxidative stress because of low levels of free-radical quenchingenzymes. Thus, there is a need to increase the number of viableinsulin-secreting cells as a means for improving tissue or celltransplantation or treating type 1 diabetes mellitus.

SUMMARY

Provided herein in certain embodiments are methods of increasing β-cellviability in a target islet. The methods may include contacting thetarget islet with a delivery vehicle including a molecule such as aGSTA4 polypeptide having at least 95% sequence identity with an aminoacid sequence of SEQ ID NO: 4 or a GSTA4 polynucleotide which encodesthe GSTA4 polypeptide. In certain embodiments, contacting the targetislet with the delivery vehicle may occur in the media or buffersolution used for isolation, preparation, or storage of the targetislet. In certain embodiments, the delivery vehicle may be a liposome,nanoparticle, nanotube, non-liposomal lipid, or polymer and may includethe GSTA4 polypeptide. In certain embodiments, the delivery vehicle mayfurther include a RLIP76 polypeptide having at least 95% sequenceidentity with an amino acid sequence of SEQ ID NO: 2. In certainembodiments, the delivery vehicle may be a plasmid or a viral vector andmay include the GSTA4 polynucleotide, the GSTA4 polynucleotide having atleast 95% sequence identity with a DNA sequence of SEQ ID NO: 3. Incertain embodiments, the delivery vehicle may further include a RLIP76polynucleotide having at least 95% sequence identity with a DNA sequenceof SEQ ID NO: 1. In certain embodiments, the delivery vehicle may be theviral vector comprising an adenovirus vector, an adeno-associated virusvector, a herpes simplex virus vector, a retrovirus vector, or alentivirus vector.

Also provided herein in certain embodiments are methods of treating adisease or condition in a subject. In certain embodiments, the methodsmay include steps such as contacting a target islet with a deliveryvehicle comprising a molecule comprising a GSTA4 polypeptide having atleast 95% sequence identity with an amino acid sequence of SEQ ID NO: 4or a GSTA4 polynucleotide which encodes the GSTA4 polypeptide andtransplanting the target islet into the subject to treat the disease orcondition. In certain embodiments, the disease or condition may be type1 diabetes. In certain embodiments, contacting the target islet with thedelivery vehicle may occur in the media or buffer solution used forisolation, preparation, or storage of the target islet. In certainembodiments, the delivery vehicle may be a liposome, nanoparticle,nanotube, non-liposomal lipid, or polymer and may include the GSTA4polypeptide. In certain embodiments, the delivery vehicle may furtherinclude a RLIP76 polypeptide having at least 95% sequence identity withan amino acid sequence of SEQ ID NO: 2. In certain embodiments, thedelivery vehicle may be a plasmid or a viral vector and may include theGSTA4 polynucleotide, the GSTA4 polynucleotide having at least 95%sequence identity with a DNA sequence of SEQ ID NO: 3. In certainembodiments, the delivery vehicle may further include a RLIP76polynucleotide having at least 95% sequence identity with a DNA sequenceof SEQ ID NO: 1. In certain embodiments, the delivery vehicle may be theviral vector comprising an adenovirus vector, an adeno-associated virusvector, a herpes simplex virus vector, a retrovirus vector, or alentivirus vector.

Also provided herein in certain embodiments are kits to increase β-cellviability in a target islet. In certain embodiments, the kits mayinclude a delivery vehicle and a molecule including a GSTA4 polypeptidehaving at least 95% sequence identity with an amino acid sequence of SEQID NO: 4 or a GSTA4 polynucleotide which encodes the GSTA4 polypeptide.In certain embodiments, the kit may include the GSTA4 polypeptide andmay further include a RLIP76 polypeptide having at least 95% sequenceidentity with an amino acid sequence of SEQ ID NO: 2. In certainembodiments, the kit may include the GSTA4 polynucleotide and mayfurther include a RLIP76 polynucleotide having at least 95% sequenceidentity with a DNA sequence of SEQ ID NO: 1. In certain embodiments,the target islet may be transplanted into a subject. In certainembodiments, the delivery vehicle may include a viral vector, plasmid,liposome, nanoparticle, nanotube, non-liposomal lipid, or polymer.

Provided herein in certain embodiments are methods of increasing β-cellviability in a target islet. The methods may include contacting thetarget islet with a delivery vehicle including a molecule such as aRLIP76 polypeptide having at least 95% sequence identity with an aminoacid sequence of SEQ ID NO: 2 or a RLIP76 polynucleotide which encodesthe RLIP76 polypeptide. In certain embodiments, contacting the targetislet with the delivery vehicle may occur in the media or buffersolution used for isolation, preparation, or storage of the targetislet. In certain embodiments, the delivery vehicle may be a liposome,nanoparticle, nanotube, non-liposomal lipid, or polymer and may includethe RLIP76 polypeptide. In certain embodiments, the delivery vehicle mayfurther include a GSTA4 polypeptide having at least 95% sequenceidentity with an amino acid sequence of SEQ ID NO: 4. In certainembodiments, the delivery vehicle may be a plasmid or a viral vector andmay include the RLIP76 polynucleotide, the RLIP76 polynucleotide havingat least 95% sequence identity with a DNA sequence of SEQ ID NO: 1. Incertain embodiments, the delivery vehicle may further include a GSTA4polynucleotide having at least 95% sequence identity with a DNA sequenceof SEQ ID NO: 3. In certain embodiments, the delivery vehicle may be theviral vector comprising an adenovirus vector, an adeno-associated virusvector, a herpes simplex virus vector, a retrovirus vector, or alentivirus vector.

Also provided herein in certain embodiments are methods of treating adisease or condition in a subject. In certain embodiments, the methodsmay include steps such as contacting a target islet with a deliveryvehicle comprising a molecule comprising a RLIP76 polypeptide having atleast 95% sequence identity with an amino acid sequence of SEQ ID NO: 2or a RLIP76 polynucleotide which encodes the RLIP76 polypeptide andtransplanting the target islet into the subject to treat the disease orcondition. In certain embodiments, the disease or condition may be type1 diabetes. In certain embodiments, contacting the target islet with thedelivery vehicle may occur in the media or buffer solution used forisolation, preparation, or storage of the target islet. In certainembodiments, the delivery vehicle may be a liposome, nanoparticle,nanotube, non-liposomal lipid, or polymer and comprises the RLIP76polypeptide. In certain embodiments, the delivery vehicle may furtherinclude a GSTA4 polypeptide having at least 95% sequence identity withan amino acid sequence of SEQ ID NO: 4. In certain embodiments, thedelivery vehicle may be a plasmid or a viral vector and may include theRLIP76 polynucleotide, the RLIP76 polynucleotide having at least 95%sequence identity with a DNA sequence of SEQ ID NO: 1. In certainembodiments, the delivery vehicle may further include a GSTA4polynucleotide having at least 95% sequence identity with a DNA sequenceof SEQ ID NO: 3. In certain embodiments, the delivery vehicle may be theviral vector comprising an adenovirus vector, an adeno-associated virusvector, a herpes simplex virus vector, a retrovirus vector, or alentivirus vector.

Also provided herein in certain embodiments are kits to increase β-cellviability in a target islet. In certain embodiments, the kits mayinclude a delivery vehicle and a molecule including a RLIP76 polypeptidehaving at least 95% sequence identity with an amino acid sequence of SEQID NO: 2 or a RLIP76 polynucleotide which encodes the RLIP76polypeptide. In certain embodiments, the kit may include the RLIP76polypeptide and may further include a GSTA4 polypeptide having at least95% sequence identity with an amino acid sequence of SEQ ID NO: 4. Incertain embodiments, the kit may include the RLIP76 polynucleotide andmay further include a GSTA4 polynucleotide having at least 95% sequenceidentity with a DNA sequence of SEQ ID NO: 3. In certain embodiments,the target islet may be transplanted into a subject. In certainembodiments, the delivery vehicle may include a viral vector, plasmid,liposome, nanoparticle, nanotube, non-liposomal lipid, or polymer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A: Blood glucose levels in C57BL-6 and glutathione S-transferaseA4 isoenzyme (GSTA4) knockout mice. Results of blood glucose (fasting)by tail vein blood from one separate determination is shown.

FIG. 1B: Blood glucose levels in C57BL-6 and glutathione S-transferaseA4 isoenzyme (GSTA4) knockout mice. Results of blood glucose (fasting)by tail vein blood from one separate determination is shown.

FIG. 1C. Serum lipids for the wild-type (C57Bl-6, black bar) and GSTA4knockout (GSTA4(−/−), grey) mice.

FIG. 1D. Serum insulin level for the wild-type (C57Bl-6) and GSTA4knockout (GSTA4(−/−)) mice.

FIG. 2: Insulin levels and the pancreatic β-cell mass in C57BL-6 andGSTA knockout mice. Pancreas were stained for hematoxylin and eosin(H&E). The % of β-cell values for n=3 is presented in the bar graph.

FIG. 3: Pancreatic histology of GSTA4 knockout mice. Formalin fixedpancreas from C57BL-6 and GSTA4 knockout mice were sectioned and stainedfor H&E.

FIG. 4A: Serum liver enzymes for C57BL-6 and GSTA4 knockout mice. Serumlevels of AST are presented.

FIG. 4B. Serum liver enzymes for C57BL-6 and GSTA4 knockout mice. Serumlevels of ALT are presented.

FIG. 5: The expression of inflammation marker genes, IL-6, TNF-alpha,MCP-1, for C57BL-6 (black) and GSTA4 knockout mice (grey).

FIG. 6A: Expression of GSTA4 or RaI-binding protein-1 (RALBP1 geneproduct, also known as RaI-interacting protein (RLIP76)) in control,empty vector (pcDNA3.1) and GSTA4 (GSTA4/pcDNA3.1) transfected INS-1cells. Several G418-resistant stable clones expressing GSTA4 or RLIP76were characterized by RT-PCR.

FIG. 6B: A bar graph showing the fold change of the RT-PCR productsdescribed in FIG. 6A.

FIG. 6C: Gene specific primers were used for reverse transcription usingRT kit (Applied Biosystems).

FIG. 6D: Immunocytochemistry showing INS-1 cells transfected with eithercontrol, empty vector (pcDNA3.1) or GSTA4 (GSTA4/pcDNA3.1).

FIG. 7A: Expression of GSTA4 and insulin in INS-1 cells. A.Immunocytochemistry showing DAPI, GSTA4, and insulin staining in cellstransfected with control, vector, or GSTA4.

FIG. 7B: Expression of insulin in INS-1 cells. A bar graph showing themean insulin intensity of cells. The values of n=3 are presented.

FIG. 8: Expression of genes involved in insulin signaling in control andGSTA4 transfected cells. A bar graph showing the fold change ofexpression of genes involved in insulin signaling as determined byRT-PCR. The left bar (medium grey) represents data from the control, themiddle bar (dark grey) represents gene expression from clone D2expressing GSTA4, and the right bar (light grey) represents geneexpression from clone D4 expressing GSTA4.

FIG. 9: GSTA4 overexpression protects INS-1 cells from oxidative stress.A schematic shows the process of treating cells with H₂O₂ to induceoxidative stress. Fluorescence of cells is shown after treatment withH₂O₂ as described.

FIG. 10: Effect of GSTA4 transfection on proliferation of INS-1 cells.Flow cytometry data shows the overexpression of GSTA4 prevents the cellsfrom H₂O₂ induced oxidative stress (see trace labeled C).

FIG. 11: Effect of GSTA4 and RLIP76 transfection on protection of INS-1cells from H₂O₂ induced cell apoptosis by flow cytometry. Representativeresults for one of three independent measurements are presented. Thestatistical comparison showed that the difference in apoptotic fractionwas significant for both enzymes (vector vs. GSTA4 transfected, p<0.01and vector vs. RLIP76 transfected cell, p<0.01).

FIG. 12: Effect of GSTA4 or RLIP76 transfection on proliferation ofINS-1 cells. A bar graph shows the cell proliferation results of a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium Bromide (MTT)assay. Fold change was normalized to control cells.

FIG. 13A: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. FIG. 13A shows cells transfected with control.

FIG. 13B: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. FIG. 13B shows cells transfected with control.

FIG. 13C: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. FIG. 13C shows cells transfected with GSTA4.

FIG. 13D: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. FIG. 13D shows cells transfected with GSTA4.

FIG. 13E: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. FIG. 13E shows cells transfected with RLIP76.

FIG. 13F: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. FIG. 13F shows cells transfected with RLIP76.

FIG. 13G: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. After 25 days, the RLIP76-transfected islets weredissociated by trypsinization and grown in culture medium containing 11mM glucose and 100 μg/mL G418. Cell morphology was determined by phasecontrast microscopy (note cell division in G).

FIG. 13H: Effect of MaP enzyme transfection on human pancreatic isletcultures. The change of morphology at day 20 of the islets is shown at20× magnification. After 25 days, the RLIP76-transfected islets weredissociated by trypsinization and grown in culture medium containing 11mM glucose and 100 μg/mL G418. Cell morphology was determined by phasecontrast microscopy.

FIG. 13I(A): A gel showing the RT-PCR results of the expression ofRLIP76 in the RLIP76-transfected dissociated cells using two pair ofgene specific primers (see FIG. 21 for primers).

FIG. 13I(B). Western blot analysis of the RLIP76-transfected dissociatedcells.

FIG. 14: Effect of RLIP76 over-expression on proliferation of humanislet cells in-vitro. Photomicrographs of cultured islet cells taken at5 days after transient transfection of pcDNA-3 eukaryotic plasmid vectorwithout or with full-length RLIP76.

FIG. 15: Effect of RLIP76-liposomes on proliferation of human isletcells in-vitro. Photomicrographs at 20× magnification of human isletsisolated from human cadaveric pancreas that treated withRLIP76-liposomes at 40 μg/mL at day 5 and day 10.

FIG. 16: DNA sequence of RLIP76 (SEQ ID NO: 1) and amino acid sequenceof RLIP76 (SEQ ID NO: 2).

FIG. 17: DNA sequence of GSTA4 (SEQ ID NO: 3) and amino acid sequence ofGSTA4 (SEQ ID NO: 4).

FIG. 18: Messenger RNA (mRNA) sequence of Homo sapiens ralA bindingprotein 1 (SEQ ID NO: 5).

FIG. 19: Table of the gene specific primers used for RT-PCR to determineexpression of genes involved in insulin signaling in control and GSTA4transfected cells (see FIG. 8 for data from RT-PCR).

FIG. 20: Table of mouse gene-specific primers used for RT-PCR todetermine expression of inflammatory genes (see FIG. 5 for data fromRT-PCR).

FIG. 21: Table of RLIP76 gene-specific primers used for RT-PCR todetermine RLIP76 expression in dissociated cells (see FIG. 13IA forRT-PCR data). The forward primer for RLIP76 was used in combination withthe RLIP76 Reverse (497 bp size band) or the RLIP76 Reverse (894 bp sizeband) primer.

DETAILED DESCRIPTION

The following description is merely intended to illustrate variousembodiments of the invention. As such, the specific embodimentsdiscussed are not to be construed as limitations on the scope of theinvention. It will be apparent to one skilled in the art that variousequivalents, changes, and modifications may be made without departingfrom the scope of invention, and it is understood that such equivalentembodiments are to be included herein. Further, all references cited inthe disclosure are hereby incorporated by reference in their entirety,as if fully set forth herein.

According to certain embodiments, methods, compositions, and kits areprovided herein to increase islet cell viability and to treat a diseaseor condition caused by the destruction of β-cells in islets, such astype 1 diabetes mellitus. In certain embodiments, islet cells may beβ-cells, which are cells in the pancreas located in the islets ofLangerhans that function to store and release insulin. In certainembodiments, the methods described herein may include increasing thequantity of enzymes of the mercapturic acid pathway (MaP), such asRaI-binding protein-1 (RALBP1 gene product, also known asRaI-interacting protein (RLIP)), glutathione S-transferase A4 isoenzyme(GSTA4), or a combination thereof, in islets. RLIP refers to allsplice-variants of the protein-product encoded by human RALBP1including, but not limited to, the predominant 76 kDa splice variantknown as RLIP76.

As shown in the Example below, increasing the quantity of RLIP76 orGSTA4 in islets may result in an increase in the ability of such isletsto survive in-vitro prior to transplantation and to survive in humans inwhom islets are transplanted. In certain embodiments, the quantity ofRLIP76 polypeptides and/or GSTA4 polypeptides in islets may be increasedthrough delivery of RLIP76 polypeptides and/or GSTA4 polypeptides asdescribed herein or RLIP76 polynucleotides and/or GSTA4 polynucleotidesas described herein to the islets.

MaP enzymes function as defenses against oxidative stress. The examplesprovided herein demonstrate that augmenting the activity of the MaP byincreasing the MaP enzymes including RLIP76 or human GSTA4 leads to anincrease in the viability and insulin-secreting ability of pancreaticislet cells. The MaP has been shown to be the primary defense againstoxidative stress and electrophilic lipid alkenals, such as4-OH-t-2-nonenal (4HNE), generated during oxidative stress andmetabolized primarily to glutathione-electrophile conjugates. GSTA4 andRLIP76 are the two major determinants of 4HNE levels in cells.Furthermore, RLIP76 is the rate limiting enzyme for both MaPs andclathrin dependent endocytosis which regulates the signaling of insulinand other peptide-hormones. Over-expression of GSTA4 has shown toincrease the proliferation of normal epithelial cells to >50%.Therefore, targeting GSTA4 and RLIP76 to increase survival andproliferation of β-cells may have clinical utility in preserving andmaintaining functional β-cell mass in early onset type 1 diabetics andin protecting newly formed or regenerated β-cells from destruction andproviding functional β-cells for islet transplantation. The resultsprovided herein demonstrate improved survival and reduced apoptosis inislet cells augmented with RLIP76 or GSTA4. Characterization of β-cellsfor cell mass and insulin sensitivity in GSTA4 and RLIP76 knockout miceclearly showed the central role of these enzymes in insulin-sensitivityand β-cell survival.

The MaP utilizes glutathione (GSH) for biotransformation of chemicalsinto mercapturic acids that are excreted in the urine. The MaP has longbeen known to play a key role in metabolism and excretion of mutagenicelectrophilic chemicals (deficient in a pair of electrons) derived fromexogenous sources (poisons, xenobiotics) and metabolites of xenobiotics(benzo[a]pyrene) or drugs (acetaminophen) (Jakoby 1978; Awasthi 1994).The importance of this pathway in the metabolism and excretion ofendogenously derived electrophiles generated from the oxidativemetabolism of polyunsaturated fatty acids (PUFA) has been increasinglyrecognized more recently (Sharma 2004). Through studies over the pasttwo decades, the role of various glutathione S-transferases (thatcatalyze the first committed step of this pathway) in defending cellsfrom pro-apoptotic and mutagenic effects of PUFA-derived reactiveoxidant and electrophilic compounds has been elucidated. These studieshave shown the importance of the alpha class GST isoenzymes (GSTA) asexcellent catalysts for reductive metabolism of lipid-hydroperoxidesoriginating from PUFA (Yang 2002). Through these studies, the key roleof a specific alpha-class enzyme, GSTA4, in metabolism of the toxic andmutagenic reactive aldehydes that are byproducts of peroxidation of PUFAhas been identified. 4HNE is the predominant reactive aldehyde producedfrom peroxidation of PUFA, and is the preferred substrate for GSTA4.GSTA4 catalyzes the formation of the glutathione (GSH)-4HNE adduct whichmust be subsequently transported out of cells by RLIP76 before it can befurther metabolized to a mercapturic acid. The GSTA4 knockout mouse wascreated and showed that lack of this enzyme causes an increase in 4HNEin mouse tissues (Engle 2004). Subsequently, the major MaP pathwaytransporter, RLIP76 was identified and cloned. The knockout mouselacking RLIP76 has a much greater level of oxidative stress (Awasthi2005; Warnke 2008).

Since loss of RLIP76 in knockout mice is known to increase blood andtissue markers of oxidative stress, and oxidative stress has beenimplicated as a direct cause of type 2 diabetes, insulin-resistance andhyperlipidemia, the glycemic control and blood lipid levels in thesemice was studied. Surprisingly, instead of insulin-resistance, as wouldbe predicted from high levels of oxidative stress, markedinsulin-sensitivity was found in these knockout mice. On the basis ofthis, a novel mechanism for insulin-resistance was proposed based onstress-induced induction of RLIP76 and consequent enhanced inactivationof insulin-signaling through the clathrin-dependent endocytosis (CDE)pathway (Awasthi 2010; Singhal 2011; Singhal 2011b; Singhal 2013).Because insulin-resistance is also implicated as a pathogenic mechanismin obesity, the hypothesis that the insulin-sensitivity of the RLIP76knockout mice should cause a resistance to obesity was subsequentlytested. These studies confirmed resistance to diet induced obesity ofthese mice (Singhal 2013). As part of these studies, the effect ofRLIP76 knockout on blood insulin levels and on pancreatic islet cellswhich make and secrete insulin was investigated. Consistent with theknown sensitivity to oxidative stress of islet cells, smaller pancreaticislets and lower insulin content of the islets were found.

However, the studies from the RLIP76 knockout mice did not fully answerthe question of whether aberrant glycemic control in these mice wassimply due to the deficient function of the MaP, or due morespecifically to the loss of CDE in which RLIP76 plays a crucialcatalytic role. Thus, the experiments in Example 1 below were performedto characterize the glycemic control mechanisms in the GSTA4 knockoutmice, which are partially deficient in the MaP, but have a normal CDEmechanism.

The studies provided herein show a glycemic control mechanism in GSTA4and the effect of MaP enzymes on the viability of islet cells. This isthe first demonstration of a role of GSTA4 in regulating islet cellviability, insulin-secretion, as well as the regulation of expression ofgenes that control insulin-secretion. The studies described herein alsoprovide a novel model of type 1 diabetes.

As provided in Example 1 below, similar to the results shown previouslyin the RLIP76 knockout mouse, the GSTA4 knockout mouse also had smallerislets and reduced plasma insulin levels. Although oxidative stress isknown to be increased in both RLIP76 and GSTA4 knockout mice, theirphenotype with respect to glycemic control were quite distinct. Whereasthe RLIP76 knockout mouse is a model of insulin sensitivity(hypoglycemia despite low insulin levels), the GSTA4 knockout mice isdiabetic, with normal insulin-sensitivity, a very interesting model fortype 1 diabetes. Also, unlike the RLIP76 knockout mouse which ishypolipidemic, serum lipids in the GSTA4 knockout mouse were unaltered.The GSTA4 knockout mouse is also characterized by histological evidenceof tissue damage, elevated liver enzymes, and increased cytokines.

As shown in Example 1 below, augmenting either RLIP76 or GSTA4 incultured INS-1 cells reduced apoptosis and protected cells fromoxidative stress, which is the first demonstration of these effects ofMaP enzyme augmentation in islet cells. Thus, as provided herein,increasing the expression or quantity of these enzymes in human isletstransplanted to treat type 1 diabetes enzymes should improve theefficacy of islet cell transplantation. Results provided herein showedmarkedly improved ability of human islets to survive in-vitro cellculture upon augmentation of MaP enzymes, which indicates the potentialhuman application of this approach for improving islet celltransplantation.

The ability of MaP enzymes to induce proliferation of INS-1 cells and toincrease insulin production is very novel and of high significance. Theobservations provided herein regarding the ability of GSTA4 to causeup-regulation of insulin-regulatory transcription factor is highly noveland may be applicable in the future for other possible novelapplications. Because islet cells stably overexpressing GSTA4 may beimmortalized and continuously self-replicating, and insulin-secretion bythe islet cells could be controlled by regulating GSTA4 expression, theymay be useful to treat type 1 diabetes if they were encapsulated innano-tubes and transplanted into human with type 1 diabetes. Thesecretion of insulin by these cells could be controlled using drugs suchas tetracycline by using a tetracycline-responsive promoter upstream ofthe GSTA4 gene.

According to certain embodiments herein, methods, compositions, and kitsherein are provided to increase islet cell viability. In certainembodiments, the islet cell may be a β-cell. In certain embodiments,increasing islet cell viability includes, without limitation, increasinginsulin secretion by β-cells in islets, increasing survival of isletcells, preserving and maintaining functional β-cell mass of islets,increasing proliferation of islet cells, reducing apoptosis, and/orreducing oxidative stress in islet cells. As discussed herein andsupported by the Example below, increasing the quantity of RLIP76 orGSTA4 protein in islets to be transplanted or in transplanted islets mayresult in an increase in the ability of such islets to survive in-vitroprior to transplantation and to survive in humans in whom islets aretransplanted.

According to certain embodiments, increasing islet cell viability may beaccomplished by increasing the expression or quantity of RLIP76polypeptides, GSTA4 polypeptides, or a combination thereof in theislets. Accordingly, in certain embodiments, the expression or quantityof RLIP76 polypeptides, GSTA4 polypeptides, or a combination thereof(i.e., RLIP76 polypeptides and GSTA4 polypeptides) in islets may beincreased through delivery of RLIP76 polypeptides, GSTA4 polypeptides,or a combination thereof (i.e., RLIP76 polypeptides and GSTA4polypeptides) or RLIP76 polynucleotides, GSTA4 polynucleotides, or acombination thereof (i.e., RLIP76 polynucleotides and GSTA4polynucleotides) to the islets. It is also contemplated that bothpolynucleotides (i.e., GSTA4 and RLIP76) and polypeptides (i.e., GSTA4and RLIP76) described herein may be delivered to islets in order toincrease viability of islets.

In certain embodiments, an “islet” or “target islet” may include,without limitation, a human cadaver islet, an islet of a subject, anislet to be transplanted into a subject, or an islet transplanted into asubject. An “islet” or a “target islet” includes populations of cellswhich produce hormones in response to glucose levels, including β-cells.

RLIP76 is a multifunctional protein that is encoded in humans onchromosome 18p11.3 by a gene with 11 exons and 9 introns (see NCBIReference Sequence: NM_006788 and SEQ ID NO: 5 (FIG. 18) for the mRNAsequence of the Homo sapiens ralA binding protein 1). The proteinproduct of the gene is typically a 76 kDa protein (i.e., RLIP76);however, splice-variants including a 67 kDa polypeptide and longer 80kDa or 102 kDa polypeptides have also been identified. In certainembodiments, it is contemplated that other splice-variants may be usedin place of RLIP76 in the embodiments described herein.

According to certain embodiments herein, a RLIP76 polypeptide maycomprise, consist of, or consist essentially of the amino acid sequenceas provided in SEQ ID NO: 2 (i.e., human RLIP76 amino acid sequence,NCBI Reference Sequence: NP_006779.1, see FIG. 16). In certainembodiments, a RLIP76 polypeptide may be GMP-grade, for example, theGMP-grade RLIP76 produced in large scale by Terapio Inc., Austin, Tex.In certain embodiments, a RLIP76 polynucleotide may be anypolynucleotide that encodes an RLIP76 polypeptide as described herein.In certain embodiments, a RLIP76 polynucleotide may comprise, consistor, or consist essentially of a DNA sequence provided in SEQ ID NO: 1(i.e., RLIP76 DNA sequence, see FIG. 16). In certain embodiments, aRLIP76 polynucleotide may be a RLIP76 gene.

According to certain embodiments herein, a GSTA4 polypeptide maycomprise, consist of, or consist essentially of the amino acid sequenceas provided in SEQ ID NO: 4 (i.e., human GSTA4 amino acid sequence, NCBIReference Sequence: NP 001503.1, see FIG. 17). In certain embodiments, aGSTA4 polynucleotide may be any polynucleotide that encodes a GSTA4polypeptide as described herein. In certain embodiments, a GSTA4polynucleotide may comprise, consist or, or consist essentially of theDNA sequence provided in SEQ ID NO: 3 (i.e., GSTA4 DNA sequence, seeFIG. 17). In certain embodiments, a GSTA4 polynucleotide may be a GSTA4gene.

In certain embodiments, the polynucleotides described herein may berecombinant or non-naturally occurring polynucleotides. In certainembodiments, the polynucleotides described herein may be messenger RNA(mRNA) or DNA. In certain embodiments, the polynucleotides may be cDNA.

Polynucleotides as described herein are not limited to the functionalregion of the nucleotide sequence, and may include at least one of anexpression suppression region, a coding region, a leader sequence, anexon, an intron, and an expression cassette (see, e.g. Papadakis et al.,“Promoters and Control Elements: Designing Expression Cassettes for GeneTherapy,” Current Gene Therapy (2004), 4, 89-113). Further,polynucleotides may include double stranded DNA, single stranded DNA orRNA. The RLIP76 polynucleotides and GSTA4 polynucleotides describedherein may be fragments or mutants of the full length RLIP76polynucleotides or GSTA4 polynucleotides, respectively. A fragment meansa part of the polynucleotide that encodes a polypeptide which providessubstantially the same function as the polypeptide encoded by thefull-length polynucleotide. Examples of polynucleotide mutants includenaturally occurring allelic mutants; artificial mutants; andpolynucleotide sequences obtained by deletion, substitution, addition,and/or insertion of one or more nucleotides to the polynucleotidesequence. It should be understood that such a fragment and/or mutant ofa polynucleotide sequence encodes a polypeptide having substantially thesame function as a polypeptide encoded by the original full-lengthpolynucleotide sequence. For example, a fragment and/or mutant of aRLIP76 polynucleotide encodes a RLIP76 polypeptide that possessessubstantially the same function of a full length RLIP76 polypeptide anda fragment and/or mutant of a GSTA4 polynucleotide encodes a GSTA4polypeptide that possesses substantially the same function of a fulllength GSTA4 polypeptide.

In certain embodiments, it is contemplated that RLIP76 polypeptides andGSTA4 polypeptides as described herein may include modifications intheir amino acid sequences or chemical modifications in their structuresformulated for use with or without a delivery vehicle, such as thosedescribed herein (e.g., liposomes, nanoparticles, etc.).

According to certain embodiments herein, a polypeptide or amino acidsequence described herein may have at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,about 90%˜99.999%, about 91%˜99.999%, about 92%˜99.999%, about93%˜99.999%, about 94%˜99.999%, about 95%˜99.999%, about 96%˜99.999%,about 97%˜99.999%, about 98%˜99.999%, or about 99%˜99.999% sequenceidentity with SEQ ID NO: 2 or SEQ ID NO: 4. For example, in certainembodiments, a GSTA4 polypeptide used with the methods, compositions, orkits as described herein may have at least 95% sequence identity with anamino acid sequence of SEQ ID NO: 4. In certain embodiments, a RLIP76polypeptide used with the methods, compositions, or kits as describedherein may have at least 95% sequence identity with an amino acidsequence of SEQ ID NO: 2.

According to certain embodiments herein, a polynucleotide or DNAsequence described herein may have at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,about 90%˜99.999%, about 91%˜99.999%, about 92%˜99.999%, about93%˜99.999%, about 94%˜99.999%, about 95%˜99.999%, about 96%˜99.999%,about 97%˜99.999%, about 98%˜99.999%, or about 99%˜99.999% sequenceidentity with SEQ ID NO: 1 or SEQ ID NO: 3. For example, in certainembodiments, a GSTA4 polynucleotide used with the methods, compositions,or kits as described herein may have at least 95% sequence identity witha DNA sequence of SEQ ID NO: 3. In certain embodiments, a RLIP76polynucleotide used with the methods, compositions, or kits as describedherein may have at least 95% sequence identity with a DNA sequence ofSEQ ID NO: 1.

Codon optimization is a technique that may be used to maximize theprotein expression in an organism by increasing the translationalefficiency of the gene of interest. Different organisms often showparticular preferences for one of the several codons that encode thesame amino acid due to mutational biases and natural selection. Forexample, in fast growing microorganisms such as E. coli, optimal codonsreflect the composition of their respective genomic tRNA pool.Therefore, the codons of low frequency of an amino acid may be replacedwith codons for the same amino acid but of high frequency in the fastgrowing microorganism. Accordingly, the expression of the optimized DNAsequence is improved in the fast growing microorganism. See, e.g.http://www.guptalab.org/shubhg/pdf/shubhra_codon.pdf for an overview ofcodon optimization technology, which is incorporated herein by referencein its entirety. As provided herein, the DNA sequences described hereinmay be codon optimized for optimal polypeptide expression in aparticular type of cell line including, but not limited to, mammaliancells.

In certain embodiments, a delivery vehicle may be used to deliver aRLIP76 polynucleotide and/or GSTA4 polynucleotide to a target islet. Incertain embodiments, the delivery vehicle may include, withoutlimitation, viral vectors, plasmids, liposomes, nanoparticles,nanotubes, non-liposomal lipids, and/or polymers. In certainembodiments, the delivery vehicle may include any delivery vehicle knownto one skilled in the art for delivering polypeptides or polynucleotidesto a cell.

In certain embodiments, an increase in RLIP76 and/or GSTA4 expression ina target islet may occur through genetic or epigenetic methods, whichincludes, without limitation, partial or stable transfection ortransduction of the respective full length gene or DNA sequence into thetarget islet.

In certain embodiments, a RLIP76 polynucleotide and/or GSTA4polynucleotide may be delivered to a target islet via transfection, inwhich the RLIP76 polynucleotide and/or GSTA4 polynucleotide isintroduced into the target islet either transiently or permanently (alsocalled persistent or stable). In certain embodiments, transfection ofthe target islet may occur using non-viral transfection deliveryvehicles including, but not limited to, a plasmid, liposome,nanoparticle, nanotube, non-liposomal lipid, or polymer. For example, incertain embodiments, a tetracycline-controlled transcriptionalactivation plasmid may be used to deliver the RLIP76 polynucleotidesand/or GSTA4 polynucleotides to the target islets and further controlexpression of the transfected polynucleotides. In certain embodiments,the tetracycline-controlled transcriptional activation plasmid mayinclude a tetracycline-responsive promoter upstream of the RLIP76polynucleotides and/or GSTA4 polynucleotides in the plasmid.

In certain embodiments, a RLIP76 polynucleotide and/or GSTA4polynucleotide may be delivered to a target islet via virus-mediatedtransfection (i.e., transduction), in which the RLIP76 polynucleotideand/or GSTA4 polynucleotide is introduced into the target islet.Transduction may be either transient or permanent (also calledpersistent or stable). In certain embodiments, viral vectors may be usedto transduce a target islet. In certain embodiments, the viral vectorsmay include an adenovirus vector, an adeno-associated virus vector, aherpes simplex virus vector, a retrovirus vector, or a lentivirusvector.

In certain embodiments, a RLIP76 polypeptide and/or GSTA4 polypeptidemay be delivered to a target islet via a delivery vehicle including,without limitation, a liposome, nanoparticle, nanotube, non-liposomallipid, or polymer. For example, in certain embodiments, a RLIP76polypeptide and/or GSTA4 polypeptide may be encapsulated in liposomesand delivered to a target islet to increase the quantity of the RLIP76polypeptide and/or GSTA4 polypeptide in the target islet.

The novel findings described herein offer strong evidence for thefeasibility of using RLIP76 protein and/or GSTA4 protein as an additiveto islet preparation medium for preservation of the viability of humanislets for transplantation. In certain embodiments, delivery of a RLIP76and/or GSTA4 polypeptide or a RLIP76 and/or GSTA4 polynucleotide to atarget islet may be performed in a media or buffer used for isolation,preparation or storage of the target islets. In certain embodiments, itis also contemplated that certain chemical agents known to increasepolynucleotide or polypeptide expression of RLIP76 and/or GSTA4 may beadded to the media or buffer used for isolation, preparation or storageof islets in order to increase expression of RLIP76 and/or GSTA4.

According to certain embodiments, methods of treating a disease orcondition in a subject are also provided herein. In certain embodiments,a “disease or condition” as described herein may be, without limitation,a disease or condition caused by a decrease in islet cell viability(e.g., β-cell viability), a destruction of β-cells, and/or a decrease inβ-cell insulin secretion. In certain embodiments, a disease or conditionmay be diabetes mellitus or a peptide hormone deficiency. In certainembodiments, diabetes mellitus may be type 1 diabetes mellitus. Incertain embodiments, a disease or condition may include any disease orcondition that relates to a β-cell deficiency.

In certain embodiments, methods of treating a disease or condition in asubject may comprise delivering a RLIP76 polynucleotide and/or GSTA4polynucleotide to a target islet, and transplanting the target isletinto the subject to treat the disease or condition. In certainembodiments, methods of treating a disease or condition in a subject maycomprise delivering a RLIP76 polypeptide and/or GSTA4 polypeptide to atarget islet, and transplanting the target islet into the subject totreat the disease or condition. In certain embodiments, the RLIP76polynucleotide and/or GSTA4 polynucleotide or RLIP76 polypeptide and/orGSTA4 polypeptide may be delivered to a target islet using any of themethods and delivery vehicles as described herein. For example, incertain embodiments, the delivery vehicle used for delivering thepolypeptides or polynucleotides as described herein may include, withoutlimitation, any delivery vehicle known to one skilled in the art fordelivering polypeptides or polynucleotides to a cell including, withoutlimitation, viral vectors, plasmids, liposomes, nanoparticles,nanotubes, non-liposomal lipids, or polymers. In certain embodiments,the delivery of the RLIP76 polynucleotide and/or GSTA4 polynucleotidemay be transient or permanent. In certain embodiments, a RLIP76polynucleotide and/or GSTA4 polynucleotide or a RLIP76 polypeptideand/or GSTA4 polypeptide may be added to islet isolation, storage orpreparation media prior to transplantation of the islet into thesubject.

In certain embodiments, methods of treating a disease or condition in asubject may comprise administering a therapeutically effective amount ofa RLIP76 polynucleotide and/or GSTA4 polynucleotide or a RLIP76polypeptide and/or GSTA4 polypeptide to an islet of the subject to treatthe disease or condition. The therapeutically effective amount of aRLIP76 polynucleotide and/or GSTA4 polynucleotide or a RLIP76polypeptide and/or GSTA4 polypeptide may be administered using any ofthe delivery vehicles described herein. The treatment may be used totreat any disease or condition as described herein. The RLIP76polypeptides and/or GSTA4 polypeptides or RLIP76 polynucleotides and/orGSTA4 polynucleotides described herein may be administered alone or aspart of a composition comprising the polypeptides or polynucleotides.The compositions may also include any one or more delivery vehicles thatare described herein. The compositions may be delivered in any effectivemanner and may be delivered and/or utilized alone or in combination withanother therapy.

In certain embodiments, the methods described herein may be used withother MaP enzymes.

“Treating” or “treatment” of a disease or condition may refer topreventing the disease or condition, slowing the onset or rate ofdevelopment of the disease or condition, reducing the risk of developingthe condition, preventing or delaying the development of symptomsassociated with the disease or condition, reducing or ending symptomsassociated with the disease or condition, generating a complete orpartial regression of the disease or condition, or some combinationthereof. Treatment may also mean a prophylactic or preventativetreatment of a disease or condition.

A “subject in need thereof” as used herein with regard to a disease orcondition refers to a human subject who has previously been diagnosedwith a disease or condition, is suspected of having a disease orcondition, and/or a subject who has previously exhibited one or moresymptoms associated with a disease or condition.

The phrases “patient” and “subject” are used interchangeably herein.

The term “effective amount” as used herein refers to an amount of aRLIP76 polypeptide and/or a GSTA4 polypeptide or a RLIP76 polynucleotideand/or a GSTA4 polynucleotide described herein that produces a desiredeffect. For example, a population of cells may be contacted with aneffective amount of a RLIP76 polypeptide and/or a GSTA4 polypeptide or aRLIP76 polynucleotide and/or a GSTA4 polynucleotide described herein tostudy its effect in vitro (e.g., cell culture) or to produce a desiredtherapeutic effect ex vivo or in vitro. An effective amount of a RLIP76polypeptide and/or a GSTA4 polypeptide or a RLIP76 polynucleotide and/ora GSTA4 polynucleotide described herein may be used to produce atherapeutic effect in a subject, such as preventing or treating a targetdisease or condition, alleviating symptoms associated with the diseaseor condition, or producing a desired physiological effect. In such acase, the effective amount of a RLIP76 polypeptide and/or a GSTA4polypeptide or a RLIP76 polynucleotide and/or a GSTA4 polynucleotidedescribed herein is a “therapeutically effective amount,”“therapeutically effective concentration” or “therapeutically effectivedose.” The precise effective amount or therapeutically effective amountis an amount of the composition that will yield the most effectiveresults in terms of efficacy of treatment in a given subject orpopulation of cells. This amount will vary depending upon a variety offactors, including but not limited to the characteristics of a RLIP76polypeptide and/or a GSTA4 polypeptide or a RLIP76 polynucleotide and/ora GSTA4 polynucleotide described herein (including activity,pharmacokinetics, pharmacodynamics, and bioavailability), thephysiological condition of the subject (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosage,and type of medication) or cells, the nature of the pharmaceuticallyacceptable carrier or carriers in the formulation, and the route ofadministration. Further an effective or therapeutically effective amountmay vary depending on whether a RLIP76 polypeptide and/or GSTA4polypeptide or RLIP76 polynucleotide and/or GSTA4 polynucleotidedescribed herein is administered alone or in combination with anotherpolypeptide or polynucleotide, compound, drug, therapy or othertherapeutic method or modality. One skilled in the clinical andpharmacological arts will be able to determine an effective amount ortherapeutically effective amount through routine experimentation, namelyby monitoring a cell's or subject's response to administration of aRLIP76 polypeptide and/or GSTA4 polypeptide or a RLIP76 polynucleotideand/or a GSTA4 polynucleotide described herein and adjusting the dosageaccordingly. For additional guidance, see Remington: The Science andPractice of Pharmacy, 21^(st) Edition, Univ. of Sciences in Philadelphia(USIP), Lippincott Williams & Wilkins, Philadelphia, Pa., 2005, which ishereby incorporated by reference as if fully set forth herein.

A “pharmaceutically acceptable carrier” as used herein refers to apharmaceutically acceptable material, composition, or delivery vehiclethat is involved in delivering a RLIP76 polypeptide and/or a GSTA4polypeptide or a RLIP76 polynucleotide and/or a GSTA4 polynucleotide asdescribed herein of interest to a cell, tissue or organ (i.e.,pancreas). A pharmaceutically acceptable carrier may comprise a varietyof components, including but not limited to a liquid or solid filler,diluent, excipient, solvent, buffer, encapsulating material, surfactant,stabilizing agent, binder, or pigment, or some combination thereof. Eachcomponent of the carrier must be “pharmaceutically acceptable” in thatit must be compatible with the other ingredients of the composition andmust be suitable for contact with any cell, tissue or organ that it mayencounter, meaning that it must not carry a risk of toxicity,irritation, allergic response, immunogenicity, or any other complicationthat excessively outweighs its therapeutic benefits.

Examples of pharmaceutically acceptable carriers for use in thecompositions provided herein include, but are not limited to, (1)sugars, such as lactose, glucose, sucrose, or mannitol; (2) starches,such as corn starch and potato starch; (3) cellulose and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)talc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols such as propyleneglycol; (11) polyols such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) disintegrating agents such as agar or calcium carbonate;(14) buffering or pH adjusting agents such as magnesium hydroxide,aluminum hydroxide, sodium chloride, sodium lactate, calcium chloride,and phosphate buffer solutions; (15) alginic acid; (16) pyrogen-freewater; (17) isotonic saline; (18) Ringer's solution; (19) alcohols suchas ethyl alcohol and propane alcohol; (20) paraffin; (21) lubricants,such as talc, calcium stearate, magnesium stearate, solid polyethyleneglycol, or sodium lauryl sulfate; (22) coloring agents or pigments; (23)glidants such as colloidal silicon dioxide, talc, and starch ortri-basic calcium phosphate; (24) other non-toxic compatible substancesemployed in pharmaceutical compositions such as acetone; and (25)combinations thereof.

The term “about” as used herein means within 5% or 10% of a stated valueor range of values.

Provided herein in certain embodiments are kits for carrying out theassays and methods disclosed herein. The kits disclosed herein mayinclude a RLIP76 polypeptide and/or a GSTA4 polypeptide or a RLIP76polynucleotide and/or a GSTA4 polynucleotide as described herein. Thekits may additionally include any delivery vehicle that can be used todeliver a RLIP76 polypeptide and/or a GSTA4 polypeptide or a RLIP76polynucleotide and/or a GSTA4 polynucleotide to an islet. The kits mayadditionally include other pigments, binders, surfactants, buffers,stabilizers, and/or chemicals. In certain embodiments, the kits mayadditionally include substances that may be used for testing levels ofRLIP76 polynucleotides and/or GSTA4 polynucleotides or RLIP76polypeptides and/or GSTA4 polypeptides. In certain embodiments, the kitsmay include substances that may be used for testing insulin and glucoselevels in serum. In certain embodiments, the kits provided hereincomprise instructions in a tangible medium.

One of ordinary skill in the art will recognize that the variousembodiments described herein can be combined. For example, steps fromthe various methods of treatment disclosed herein may be combined inorder to achieve a satisfactory or improved level of treatment.

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention. It will be understood thatmany variations can be made in the procedures herein described whilestill remaining within the bounds of the present invention. It is theintention of the inventors that such variations are included within thescope of the invention.

EXAMPLES Example 1: Targeting the Mercapturic Acid Pathway to Improveβ-Cell Survival and Proliferation

Effect of GSTA4 Gene Knockout in C57BL-6 Mice.

The studies of glycemic regulation in RLIP76 gene knockout mice revealedvery unexpected findings of insulin-sensitivity, hypoglycemia,low-cholesterol and resistance to obesity, which are essentially theopposite of metabolic syndrome that would have been expected because ofa high level of tissue oxidative stress in these mice (Awasthi 2005;Warnke 2008; Awasthi 2010; Singhal 2011a; Singhal 2011b; Singhal 2013).Thus, it could not be predicted whether a GSTA4 knockout, which alsoexhibits oxidative stress (but no CDE abnormalities), would have alteredblood glucose or insulin levels, or any effects on their pancreaticislets. To address these questions, the first known studies of glycemiccontrol in GSTA4 knockout mice were performed, as created by Yang 2002.Unlike the results in RLIP76 knockout mice, the 8 week old GSTA4knockout mice were actually hyperglycemic; in addition, unlike RLIP76knockout animals that had decreased serum cholesterol and triglycerides,these lipids as well as HDL-cholesterol were unaffected in theseanimals. However, similar to what was previously seen in the RLIP76knockout mice, serum insulin was decreased in the GSTA4 knockout mice(see FIG. 1). This decrease of approximately 50% in serum insulin levelseen in GSTA4 knockout mice was less dramatic (˜75%) than what was seenin the RLIP76 knockout mice.

Taken together, the elevated glucose and low insulin level in the GSTA4knockout mouse indicated that these mice have a phenotype consistentwith type 1 diabetes. The ratio of glucose/insulin is nearly the same inwild-type and GSTA4 knockout mice, indicating that the hyperglycemia isnot due to insulin-resistance. This is of particular interest because noprevious genetic alterations (knockout or transgenic) have yielded asimilar mouse. The previous models are driven primarily by geneticalterations that lead to immune mediated pancreatic islet destructionthat occur gradually over the life-span of the mouse (20-40 weeks) ordrug-induced destruction of pancreatic islets using streptozotocin, achemotherapy drug that causes islet cell death due to oxidativestress-mediated apoptosis. The blood glucose and lipid alterations inthe GSTA4 knockout mice are novel. In addition, because GSTA4 is notinvolved in CDE and CDE is not affected in these mice, the mechanism ofdiabetes is likely related to deficient pancreatic insulin production orsecretion rather than to peripheral insulin-resistance.

The reason for decreased insulin levels was examined by studying thesize and insulin-content of the pancreatic islets. Histologicalexamination of hematoxylin-eosin slides of the pancreas showed thattheir islets were significantly smaller in the GSTA4 knockout mouse.Examination of insulin stained pancreatic tissue by fluorescencemicroscopy at low power confirmed the decreased number and size of theislets. Quantitation of insulin staining confirmed a significantlyreduced islet cell mass (FIG. 2). Higher magnification analysis ofhematoxylin-eosin stained sections of pancreatic tissues revealedsignificantly disorganized pancreatic tissue, without significantinflammatory cell infiltration (FIG. 3). Measurements of serum levels ofAST and ALT enzymes were also found to be increased in the GSTA4knockout mice (FIG. 4). Though most frequently used as markers of liverdamage, these enzymes are also a generalized measure of damage totissues, including the pancreas. Histological examination of otherorgans did not reveal significant damage, suggesting that the pancreasis particularly susceptible to the loss of GSTA4.

Because oxidative-stress or inflammation promoting cytokines secreted bythe liver have generalized pro-inflammatory effects that have been shownto adversely affect pancreatic tissue, the expression of three of thesegenes, IL-6, TNFα, and MCP1 was measured in liver tissues. Results ofthese studies showed marked elevation of IL-6 and TNFα (see FIG. 5). Incontrast MCP-1, and inflammatory and immune regulatory cytokine secretedby monocytes, macrophages and dendritic cells was not altered. Thesefindings suggest that oxidative-stress itself rather than inflammationplay a greater role in damage to the pancreas in the GSTA4 knockoutmice. These findings indicated that GSTA4 plays a significant role inprotection of the pancreas as well as pancreatic islets by reducingoxidative stress.

The Effect of Over-expression MaP Genes in Rat INS-1 Cell Line.

To examine the mechanisms through which the MaP enzymes protect isletcells, the effect of GSTA4 or RLIP76 over-expression was examined inINS-1, a rat insulinoma derived cell line which is an widely acceptedmodel for studies of islet cells because of the inability to culturenon-transformed islet-cells in cell culture models. GSTA4 or RLIP76 wereover-expressed in INS-1 cells by transfection with pcDNA3.1 eukaryoticexpression vector. Northern blot analysis of total RNA extractedconfirmed successful transfection. The Northern blots and theirdensitometric quantitation from these studies showed successfulover-expression of GSTA4 in three selected clones and of RLIP76 in oneclone (FIGS. 6A and B, respectively) (see FIG. 6C for primer sequencesused for reverse transcription). Immunohistochemical staining for GSTA4transfection confirmed increased enzyme expression (FIG. 1D).

The effect of GSTA4 over-expression of the insulin-levels in these cellswas examined by immunohistochemistry. These studies showed asignificantly increased intracellular content of insulin by fluorescencemicroscopy (FIG. 7A) and was confirmed by scanning densitometry forinsulin staining (FIG. 7B). The potential mechanism for increasedinsulin production in these cells was examined by using RT-PCR tocompare the expression of genes known to encode insulin and to regulateinsulin expression. Mice are known to have two insulin genes, bothresponsive to glucose levels; they differ because Ins-1 expression isinsensitive to oxidative-stress whereas the expression of Ins-2 issuppressed by oxidative-stress. Because GSTA4 suppressesoxidative-stress, it was predicted that Ins-1 should be up-regulated inGSTA4 over-expressing cells. This prediction was verified by results ofRT-PCR showing over-expression of Ins-1 and no effect on Ins-2 mRNA(FIG. 8, bar graph). These findings suggest that the lower levels ofinsulin in GSTA4 knockout mice may be due to oxidative stress thatresults in a differential decrease in the expression of the Ins-1 gene.The mechanism of the altered transcription may be due to increases inthe NgN3 and PDX-1 transcription factors that regulate insulin geneexpression. In contrast, glucagon expression, which is inversely relatedto insulin expression, was decreased as expected. These findings are thefirst demonstration of the ability of MaP enzymes to regulate insulincontent of islet cells, the differential increase in Ins-1 expressiondue to suppression of oxidative-stress by GSTA4 and that increases inGSTA4 may affect insulin expression through effects on PDX-1 and NgN3.

Effect of Gene Over-Expression on Cellular Oxidative Stress.

Pancreatic islets are particularly susceptible to oxidative stress, anddrugs that cause oxidative stress are known to ablate pancreatic isletsin rats and mice, rendering them diabetic. The mercapturic acid pathwayis known to reduce cellular oxidative stress. To ensure that theoverexpression of these enzymes did indeed have this known effect andthat the observed effects on insulin expression were due to this effect,the effect of oxidative stress was measured in a standard fashion, afterexposure to hydrogen-peroxide in cells with over-expression of GSTA4 orRLIP76. The fluorescent cytochemistry method that was used employed thedichloro-dihydro-fluorescein diacetate (DCFH-DA) fluorescent dye thatreacts with free-radicals. The chemical basis of this widely acceptedmethod is shown (FIG. 9, see schematic). Results of these studies showedthat both GSTA4 and RLIP76 proteins conferred significant protectionfrom oxidative stress (FIG. 9). The protection from oxidative stress byGSTA4 transfection was confirmed by flow-cytometric measurement ofoxidative-stress using DCFA-DA (FIG. 10). The effect of oxidativestress-mediated apoptosis was also examined by flow-cytometry using duallabeling with propidium iodide and annexin V. Results of these studiesshowed that hydrogen peroxide exposure caused apoptosis, that it wassuppressed by GSTA4 or RLIP76 transfection (see FIG. 11). These resultsare novel because this is the first study showing that augmentingcellular GSTA4 or RLIP76 suppresses apoptosis in islet cells. Theseresults are significant because they imply that augmenting RLIP76 orGSTA4 in islet cells could prevent apoptosis, particularly in thecontext of islet-cell transplantation where the isolation and processingof human cadaveric pancreas islet results in significant loss or viableislets due to oxidative stress.

Effect of Gene Over-Expression on Cell Proliferation.

It was previously shown that other types of cells (retinal pigmentepithelial, leukemia, lung cancer, etc.) are stimulated to proliferateupon over-expression of GSTA4 or RLIP76. To ensure that a similar effectwas present in INS-1 cells, the effects of enzyme over-expression oncell proliferation was determined by MTT assay. These resultsdemonstrated doubling of proliferation in cells overexpressing either ofthese proteins (FIG. 12). These results are novel because they are thefirst demonstration of increased cell proliferation of an islet derivedcell by over-expression of MaP enzymes.

Effect of RLIP76 on Survival of Human Pancreatic Islets in Culture.

The results showing decreased apoptosis as well as increasedproliferation are of significance because it demonstrates that it couldbe used to improve survival of human cadaveric pancreatic islet cellsduring and after isolation, prior to transplant. It is well known thatin culture, human islets (consisting of clusters of hundreds tothousands of cells) do not proliferate. Intact islets isolated fromhuman cadaveric pancreas were obtained from Department of Diabetes,Endocrinology and Metabolism, City of Hope through HDP distributioncenter, under the approved IRB (11159). The islets (˜200 IEQ) werecultured in six well plates pre-coated with HTB-9 human bladdercarcinoma cell matrix prepared as previously described (Jakoby 1994).Each plate was transfected with eukaryotic expression vector (pcDNA3.1)alone or containing RLIP76 or GSTA4 using Lipofectamine 2000transfection reagent (Invitrogen) following the manufacturersinstructions. After 5 days, the islets were selected by addition of G418(100 μg/ml) in the medium. The change in morphology of the islets wasdetermined by taking the images every day using phase contrastmicroscopy (Olympus AX50). The change of morphology at day 20 of theislets is shown at 20× magnification (FIGS. 13 A-F). After 25 days,islets transfected with RLIP76 were dissociated by trypsin-treatment andwere grown into pre-coated six well plate in culture medium containing11 mM glucose and 100 μg/mL G418. Cell morphology was determined byphase contrast microscopy. RLIP76 transfected dissociated islet cellsstarted to proliferate in culture and dividing cells were observed(FIGS. 13 G, H). The continued expression of islets in these cells wasconfirmed by RT-PCR (FIG. 13 I).

Effect of RLIP76 Over-Expression on Proliferation of Human Islet CellIn-Vitro.

Human islet cell isolated from human cadaveric pancreas (IRB#11159) weredissociated and placed in cell culture, followed 24 hours later bytransient transfection of pcDNA-3 eukaryotic plasmid vector without orwith full-length RLIP76 (see FIG. 14).

Effect of RLIP76-Liposomes on Proliferation of Human Islet CellsIn-Vitro.

Intact human islets isolated from human cadaveric pancreas (IRB#11159)were dissociated and placed in cell culture. The controls were treatedwith empty liposomes and experimental with RLIP76-liposomes at 40 μg/mL.Photomicrographs at 20× magnification of human islets isolated fromhuman cadaveric pancreas that treated with RLIP76-liposomes at 40 μg/mLat day 5 and day 10 (see FIG. 15). The photographs demonstrate continuedviability and progressive appearance of peripheral adherent cells at 5and 10 days. The islets in control cultures began to shrink and allislets had disintegrated at 5 days (data not shown).

Materials and Methods

Blood Glucose and Insulin Level in C57Bl-6 and GSTA4 Knockout Mice.

Blood was collected by heart puncture and transferred into Eppendorftubes on ice, and centrifuged at 3000×g for 10 min. The serum wascollected and the serum glucose and lipids were determined (FIG. 1).Insulin levels were determined using the “Ultrasensitive Mouse InsulinELISA” kit (Crystal Chem Inc.) following the manufacturer'sinstructions.

Insulin Level and the Pancreatic/3-Cell Mass in Control and GSTAKnockout Mice.

Formalin fixed pancreas from wild-type C57BL-6 and GSTA4 knockout micewere sectioned and stained for hematoxylin and eosin (H&E) (FIG. 2). H&Estaining was performed for the overall morphology and size of theislets. β-cell mass was determined by quantifying insulin-positive areasin nonadjacent sections at 50 μm intervals throughout the sectionsaccording to protocol using laser scanning microscope (iCys LSC, iCys3.4 software, 40× objective and 0.5 mm step 405, 488, 561 and 630laser). Contour was based on DAPI stained nuclei and peripheral maxand/or max pixel intensity.

Pancreatic Histology of GSTA4 Knockout Mice.

Formalin fixed pancreas from C57BL-6 and GSTA4 knockout mice weresectioned and stained for hematoxylin and eosin (H&E) (FIG. 3). Theslides were viewed under Olympus BX51 microscope at pictures at 20×resolution are presented. H&E staining was performed for the overallmorphology and size of the islets.

Serum Liver Enzymes in GSTA4 Knockout Mice.

Blood was collected by heart puncture and transferred into Eppendorftubes on ice, centrifuged at 3000×g for 10 min, and serum was separatedfrom the blood cells. The serum level of AST (FIG. 4A) and ALT (FIG. 4B)was performed.

Expression of Inflammation Marker Genes.

The expression of inflammatory genes was determined by RT-PCR usingmouse gene-specific primers (FIG. 5). Briefly, 1 μg of total RNA fromliver tissue was used to synthesize cDNA by reverse transcription usingRT kit (Applied Biosystems). The primers for RT-PCR are provided in FIG.20.

Expression of GSTA4 or RLIP76 in Control, Empty Vector (pcDNA3.1) andGSTA4 (GSTA4/pcDNA3.1) Transfected INS-1 Cells.

INS-1 cells at a density of 1×10⁵ cells per well were seeded in 12 welltissue culture plate. At >70% confluence, cells were transfected withpcDNA3.1 vector alone (1 μg/well) or pcDNA3.1 vector containing openreading frame (ORF) of the hGSTA4 sequence (GSTA4/pcDNA3.1), or RLIP76(RLIP76/pcDNA3.1) using Lipofectamine-2000 transfection reagent as perthe manufacturer's instructions. Stable transfectant cells were isolatedby selection on 200 μg/mL G418 for 2 weeks. Single clones of stablytransfected cells were obtained by limited dilution. Furthercharacterization of the several G418-resistant stable clones expressingGSTA4 or RLIP76 was achieved by RT-PCR (FIGS. 6A and B, respectively)and immunocytochemistry (FIG. 6D). RNA prepared using Trizol-reagent(Invitrogen) was quantified and purity determined by measuringabsorbance at 260 and 280 nm using a nano-drop spectrophotometer (ThermoScientific). Gene specific primers (FIG. 6C) were used for reversetranscription using RT kit (Applied Biosystems).

Expression of GSTA4 and Insulin in INS-1 Cells.

Expression of insulin was determined in INS-1 cells (control) and GSTA4transfected by immunocytochemistry using anti-rat GSTA4 (raised inchicken) and anti-insulin (raised in guinea pig) IgG. DAPI was used as anuclear stain (FIG. 7A). The slides were also stained and scanned forinsulin content (FIG. 7B) using laser scanning microscope (iCys LSC,iCys 3.4 software, 40× objective and 0.5 mm step 405, 488, 561 and 630laser). Contour based on DAPI stained nuclei and peripheral max and/ormax pixel intensity.

Expression of Genes Involved in Insulin Signaling in Control and GSTA4Transfected Cells.

Expression of genes involved in insulin signaling was determined byRT-PCR using gene specific primers as shown in the Table in FIG. 19.Expression of genes was determined by RT-PCR and quantified bydensitometry using Alpha Imager (see FIG. 8).

GSTA4 Overexpression Protects INS-1 Cells from Oxidative Stress.

INS-1 cells: control, or stably transfected with empty vector(pcDNA3.1), GSTA4 (GSTA4/pcDN3.1) or RLIP76 (RLIP76/pcDNA3.1) were grownon cover-slips and incubated with 1 mM DFCH-DA at 37° C. in a CO₂incubator. After 60 min, the cells were treated with 100 μM H₂O₂ andwere incubated for 30 min. Cells were washed 2 times with PBS andcover-slips were mounted and observed under a fluorescence microscope(Olympus BX51) using excitation and emission wavelengths of 480 nm and530 nm, respectively (FIG. 9).

Effect of GSTA4 Transfection on Proliferation of INS-1 Cells.

CellTrace™ CFSE cell proliferation kit was used for staining the cellsand analyzed by flow cytometry. Briefly, 1×10⁶ cells were incubated for15 min with 20 μM CSFE in complete medium in CO₂ incubator at 37° C.,washed 2 times with pre-warmed media and grown for 72 hours in standardculture media. Cells were harvested and analyzed by flow cytometryfollowing manufacturer instructions (Life Technologies).Dichloro-dihydro-fluorescein diacetate (DCFH-DA) was used for thequantification of oxidative stress in H₂O₂ treated control andtransfected cells (FIG. 10).

Effect of GSTA4 and RLIP76 Transfection on Protection of INS-1 Cellsfrom H₂O₂ Induced Cell Apoptosis by Flow Cytometry.

INS-1 cells (1×10⁶ cells/ml) were grown in a 6-well plate and treatedwith 100 μM H₂O₂ for 30 min, harvested and centrifuged at 1500×g for 5min. Cells were washed with PBS and resuspended in 400 μl of coldannexin binding buffer containing 5 μL of Annexin V-FITC (BDBiosciences) and 5 μL of 0.1 mg/mL propidium iodide. Cells wereincubated at room temperature for 10 min in the dark and were analyzedby flow cytometry. Results were processed using FloJo analysis software(FIG. 11).

Effect of GSTA4 or RLIP76 Transfection on Proliferation of INS-1 Cells.

Cell proliferation was determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium Bromide (MTT)assay as described before. Briefly, 50,000 cells were plated in 24 welltissue culture treated plate and grown in standard culture media. After48 h incubation, 20 μL of a stock solution of MTT (5 mg/mL in PBS) wasadded to each well and the plates were incubated for additional 4 h at37° C., centrifuged, and the medium was decanted. Cells weresubsequently dissolved in 100 μL DMSO with gentle shaking for 2 h atroom temperature, followed by measuring absorbance at 570 nm in a microplate reader (Tecan Infinite 200 Pro). Fold change was normalized tocontrol cells (FIG. 12).

Effect of MaP Enzyme Transfection on Human Pancreatic Islet Cultures.

The islets (˜200 IEQ) were cultured in six well plates pre-coated withHTB-9 human bladder carcinoma cell matrix prepared as previouslydescribed (Jakoby 1978). Each plate was transfected with eukaryoticexpression vector (pcDNA3.1) alone or containing RLIP76 or GSTA4 usingLipofectamine 2000 transfection reagent (Invitrogen) following themanufacturer's instructions. After 5 days, the islets were selected byaddition of G418 (100 μg/ml) in the medium. The change in morphology ofthe islets was determined by taking the images every day using phasecontrast microscopy (Olympus AX50). The change of morphology at day 20of the islets is shown at 20× magnification: Controls (FIG. 13A and FIG.13B), GSTA4 transfected (FIG. 13C and FIG. 13D) and RLIP76-transfected(FIG. 13E and FIG. 13F). After 25 days, the RLIP76-transfected isletswere dissociated by trypsinization and grown into precoated six wellplates in culture medium containing 11 mM glucose and 100 μg/mL G418.Cell morphology was determined by phase contrast microscopy (FIG. 13Gand FIG. 13E, note cell division in FIG. 13G). The expression of RLIP76was checked in these dissociated cells RT-PCR using two pairs of genespecific primers (see FIG. 13I(A) and FIG. 21 for gene specific primers)and western blot analysis (see FIG. 13I(B), using anti-RLIP76 IgG fromCell Signaling as described (Awasthi 1994).

Effect of RLIP76 Over-Expression on Proliferation of Human Islet CellIn-Vitro.

Human islet cell isolated from human cadaveric pancreas (IRB#11159) weredissociated and placed in cell culture, followed 24 hours later bytransient transfection of pcDNA-3 eukaryotic plasmid vector without orwith full-length RLIP76. Photomicrographs of cultured cells were takenat 5 days (FIG. 14).

Effect of RLIP76-Liposomes on Proliferation of Human Islet CellsIn-Vitro.

Intact human islets isolated from human cadaveric pancreas (IRB#11159)were dissociated and placed in cell culture. The controls were treatedwith empty liposomes and experimental with RLIP76-liposomes at 40 μg/mL(FIG. 15).

REFERENCES

The references, patents and published patent applications listed below,and all references cited in the specification above are herebyincorporated by reference in their entirety, as if fully set forthherein.

-   1. Jakoby W B (1978) The glutathione S-transferases: a group of    multifunctional detoxification proteins. Adv Enzymol Relat Areas Mol    Biol 46:383-414.-   2. Awasthi Y C, Sharma R, Singhal S S (1994) Human glutathione    S-transferases. Int J Biochem 26:295-308.-   3. Sharma R, Yang Y, Sharma A, Awasthi S, Awasthi Y C (2004)    Antioxidant role of glutathione S-transferases: protection against    oxidant toxicity and regulation of stress-mediated apoptosis.    Antioxid Redox Signal 6:289-300.-   4. Yang Y, Sharma R, Zimniak P, Awasthi Y C (2002) Role of alpha    class glutathione Stransferases as antioxidant enzymes in rodent    tissues. Toxicol Appl Pharmacol 182:105-115.-   5. Engle M R, Singh S P, Czernik P J, Gaddy D, Montague D C, Ceci J    D, Yang Y, Awasthi S, Awasthi Y C, Zimniak P (2004). Physiological    role of mGSTA4-4, a glutathione S-transferase metabolizing    4-hydroxynonenal: generation and studies of mGSTA4 null mouse.    Toxicol Appl Pharmacol, 194: 296-308.-   6. Awasthi S, Singhal S S, Yadav S, Singhal J, Drake K, Nadkar A,    Zajac E, Wickramarachchi D, Rowe N, Yacoub A, Boor P, Dwivedi S,    Dent P, Jarman W, John B, Awasthi Y C (2005). RALBP1 is a major    determinant of radiation sensitivity. Cancer Res, 65: 6022-6028.-   7. Warnke M M, Wanigasekara E, Singhal S S, Singhal J, Awasthi, S,    Armstrong D W (2008). The determination of    glutathione-4-hydroxynonenal (GS-HNE), E-4-hydroxynonenal (HNE), and    E-1-hydroxynon-2-en-4-one (HNO) in mouse liver tissue by LC-ESI-MS.    Analyt Bioanal Chem, 392:1325-33.-   8. Awasthi S, Singhal S S, Yadav S, Singhal J, Vatsyayan R, Zajac E,    Luchowski R, Borvak J, Gryczynski K, Awasthi Y C. (2010). A central    role of RLIP76 in regulation of glycemic control. Diabetes, March;    59(3): 714-725.-   9. Singhal S S, Wickramarachchi D, Yadav S, Leake K, Vatsyayan R,    Lelsani P, Chaudhary P, Suzuki S, Awasthi Y C, Awasthi S (2011a).    Glutathione-Conjugate Transport by RLIP76 is required for    Clathrin-Dependent Endocytosis and Chemical Carcinogenesis. Mol    Cancer Ther, 10(1):16-28.-   10. Singhal J, Nagaprashantha L, Vatsyayan R, Awasthi S, Singhal S S    (2011b). RLIP76, a glutathione-conjugate transporter, plays a major    role in the pathogenesis of metabolic syndrome. PLoS ONE, 6(9):    e24688.-   11. Singhal S S, Figarola J, Singhal J, Reddy M A, Liu X, Berz D,    Natarajan R, Awasthi S (2013). RLIP76 Protein Knockdown Attenuates    Obesity Due to a High-fat Diet. J Biol Chem. 288(32):23394-406.

1-20. (canceled)
 21. A method of increasing β-cell viability in a targetislet comprising contacting the target islet with a delivery vehiclecomprising a molecule comprising a RLIP76 polypeptide having at least95% sequence identity with an amino acid sequence of SEQ ID NO: 2 or anRLIP76 polynucleotide which encodes the RLIP76 polypeptide.
 22. Themethod of claim 21, wherein contacting the target islet with thedelivery vehicle occurs in the media or buffer solution used forisolation, preparation, or storage of the target islet.
 23. The methodof claim 22, wherein the delivery vehicle is a liposome, nanoparticle,nanotube, non-liposomal lipid, or polymer and comprises the RLIP76polypeptide.
 24. The method of claim 23, wherein the delivery vehiclefurther comprises a GSTA4 polypeptide having at least 95% sequenceidentity with an amino acid sequence of SEQ ID NO:
 4. 25. The method ofclaim 22, wherein the delivery vehicle is a plasmid or a viral vectorand comprises the RLIP76 polynucleotide, the RLIP76 polynucleotidehaving at least 95% sequence identity with a DNA sequence of SEQ IDNO:
 1. 26. The method of claim 25, wherein the delivery vehicle furthercomprises a GSTA4 polynucleotide having at least 95% sequence identitywith a DNA sequence of SEQ ID NO:
 3. 27. The method of claim 26, whereinthe delivery vehicle is the viral vector comprising an adenovirusvector, an adeno-associated virus vector, a herpes simplex virus vector,a retrovirus vector, or a lentivirus vector.
 28. A method of treating adisease or condition in a subject comprising: contacting a target isletwith a delivery vehicle comprising a molecule comprising a RLIP76polypeptide having at least 95% sequence identity with an amino acidsequence of SEQ ID NO: 2 or a RLIP76 polynucleotide which encodes theRLIP76 polypeptide and transplanting the target islet into the subjectto treat the disease or condition.
 29. The method of claim 28, whereinthe disease or condition is type 1 diabetes.
 30. The method of claim 28,wherein contacting the target islet with the delivery vehicle occurs inthe media or buffer solution used for isolation, preparation, or storageof the target islet.
 31. The method of claim 30, wherein the deliveryvehicle is a liposome, nanoparticle, nanotube, non-liposomal lipid, orpolymer and comprises the RLIP76 polypeptide.
 32. The method of claim31, wherein the delivery vehicle further comprises a GSTA4 polypeptidehaving at least 95% sequence identity with an amino acid sequence of SEQID NO:
 4. 33. The method of claim 30, wherein the delivery vehicle is aplasmid or a viral vector and comprises the RLIP76 polynucleotide, theRLIP76 polynucleotide having at least 95% sequence identity with a DNAsequence of SEQ ID NO:
 1. 34. The method of claim 33, wherein thedelivery vehicle further comprises a GSTA4 polynucleotide having atleast 95% sequence identity with a DNA sequence of SEQ ID NO:
 3. 35. Themethod of claim 34, wherein the delivery vehicle is the viral vectorcomprising an adenovirus vector, an adeno-associated virus vector, aherpes simplex virus vector, a retrovirus vector, or a lentivirusvector.
 36. A kit to increase β-cell viability in a target isletcomprising a delivery vehicle and a molecule comprising a RLIP76polypeptide having at least 95% sequence identity with an amino acidsequence of SEQ ID NO: 2 or a RLIP76 polynucleotide which encodes theRLIP76 polypeptide.
 37. The kit of claim 36, wherein the kit comprisesthe RLIP76 polypeptide and further comprises a GSTA4 polypeptide havingat least 95% sequence identity with an amino acid sequence of SEQ ID NO:4.
 38. The kit of claim 36, wherein the kit comprises the RLIP76polynucleotide and further comprises a GSTA4 polynucleotide having atleast 95% sequence identity with a DNA sequence of SEQ ID NO:
 3. 39. Thekit of claim 36, wherein the target islet is to be transplanted into asubject.
 40. The kit of claim 36, wherein the delivery vehicle comprisesa viral vector, plasmid, liposome, nanoparticle, nanotube, non-liposomallipid, or polymer.